The valuable specialty chemical succinate and its derivatives have extensive industrial applications. Succinic acid is used as a raw material for food, medicine, plastics, cosmetics, and textiles, as well as in plating and waste-gas scrubbing (66). Succinic acid can serve as a feedstock for such plastic precursors as 1,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone. Further, succinic acid and BDO can be used as monomers for polyesters. If the cost of succinate can be reduced, it will become more useful as an intermediary feedstock for producing other bulk chemicals (51). Along with succinic acid, other 4-carbon dicarboxylic acids such as malic acid and fumaric acid also have feedstock potential.
Currently, succinate is produced through petrochemical processes that are expensive and can damage the environment. A high yield succinate producing bacteria would allow replacement of a petroleum product with a feedstock that uses agricultural waste. The production of succinate, malic acid, and fumaric acid from glucose, xylose, sorbitol and other “green” sources by Escherichia coli provides a low cost renewable source of chemical feedstocks. Additionally, heterologous genes are often expressed in E. coli to produce valuable compounds such as polyketides, esters, nutritional compounds, and pigments.
Metabolic engineering has the potential to considerably improve process productivity by manipulating the throughput of metabolic pathways. Specifically, manipulating enzyme levels through the amplification, addition, or reduction of a particular pathway can result in high yields of a desired product. Various genetic improvements for succinic acid production under anaerobic conditions have been described that utilize the mixed-acid fermentation pathways of E. coli. Examples include the overexpression of phosphoenolpyruvate carboxylase (pepc) from E. coli (38). The conversion of fumarate to succinate was improved by overexpressing native fumarate reductase (frd) in E. coli (16, 57). Certain enzymes are not indigenous in E. coli, but can potentially help increase succinate production. By introducing pyruvate carboxylase (pyc) from Rhizobium etli into E. coli, succinate production was enhanced (13, 14, 15). Other metabolic engineering strategies also include inactivating competing pathways of succinate. When malic enzyme was overexpressed in a host with inactivated pyruvate formate lyase (pfl) and lactate dehydrogenase (ldh) genes, succinate became the major fermentation product (45, 21). In cultures of this pfl and ldh mutant strain, there is a large pyruvate accumulation. Overexpression of malic enzyme in this mutant strain increased succinate production driven by the high pyruvate pool toward the direction of malate formation, which subsequently was converted to succinate. An inactive glucose phosphotransferase system (ptsG) in the same mutant strain (pfl− and ldh−) had also been shown to yield higher succinate production in E. coli and improve growth (8). Unfortunately, anaerobic fermentation is hampered by the limited NADH availability, poor biomass generation, slow carbon throughput, and, therefore, slow product formation.
Because of the disadvantages of anaerobic fermentation, E. coli was genetically engineered to produce succinate under aerobic conditions (29, 34). This work provides metabolically engineered succinate production systems that can operate under aerobic conditions through pathway modeling, optimization, and genetic engineering of the aerobic central metabolism. This is the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.